U.S. patent application number 17/592268 was filed with the patent office on 2022-06-09 for cytological staining compositions and uses thereof.
The applicant listed for this patent is TriPath Imaging, Inc.. Invention is credited to William Alan Fox.
Application Number | 20220178795 17/592268 |
Document ID | / |
Family ID | 1000006168239 |
Filed Date | 2022-06-09 |
United States Patent
Application |
20220178795 |
Kind Code |
A1 |
Fox; William Alan |
June 9, 2022 |
CYTOLOGICAL STAINING COMPOSITIONS AND USES THEREOF
Abstract
The present subject matter is directed to a composition for
staining cytological material comprising a cationic dye component,
a hygroscopic polyol and optionally a water-soluble solvent, water
or water-miscible solvent, methods of use of the compositions.
Advantageously, the present compositions do not require the use of
a cover slide as is required in known staining fixatives. The
compositions are able to retain cell morphology for a period of
time such that a cover slide is not required. Further, the
compositions do not contain hazardous levels of organic components.
Preferably, the compositions consist essentially of Azure C,
glycerol and optionally water. In another aspect, the present
subject matter is directed to a method of characterizing a cell
sample comprising contacting the cell sample with a composition
comprising a dye component and a hygroscopic polyol and subjecting
the sample to analysis to determine the presence or absence of
abnormal cells.
Inventors: |
Fox; William Alan; (Lake
Wylie, SC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TriPath Imaging, Inc. |
Burlington |
NC |
US |
|
|
Family ID: |
1000006168239 |
Appl. No.: |
17/592268 |
Filed: |
February 3, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14765709 |
Aug 4, 2015 |
11274997 |
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PCT/US2014/014563 |
Feb 4, 2014 |
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17592268 |
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61760889 |
Feb 5, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 1/30 20130101; G01N
2001/302 20130101 |
International
Class: |
G01N 1/30 20060101
G01N001/30 |
Claims
1.-28. (canceled)
29. A method of preparing a cytological preparation comprising:
contacting a cell sample with a cytological staining composition,
thereby staining cells in the cell sample, wherein the cytological
staining composition comprises: i. a dye component comprising one
or more water-soluble, pH neutral, cationic dyes; and ii. one or
more hygroscopic polyols present at less than about 10% v/v of the
total composition.
30. The method of claim 29, wherein cytological staining
composition comprises water, a water-soluble solvent, a
water-miscible solvent, or mixtures thereof.
31. The method of claim 29, wherein the cell sample is associated
with a substrate.
32. The method of claim 31, wherein said substrate is selected from
the group consisting of a microscope slide, a cuvette, a microtiter
tray, a cell culture tray and a coverslip.
33. The method of claim 31, the method does not comprise
associating a solid cover slide with said sample associated with
said substrate.
34. The method of claim 29, wherein the cell sample comprises
cervical cells, abnormal cells, tumor cells, cells obtained in
tissue aspiration, cells obtained in lavage collection, cells
obtained in biopsy, or mixtures thereof.
35. A method of characterizing a cytological sample comprising: a.
contacting a cell sample with a cytological staining composition,
wherein said sample is associated with a substrate, and b.
subjecting said sample to an assay to determine the presence or
absence of abnormal cells, wherein the cytological staining
composition comprises: i. a dye component comprising one or more
water-soluble, pH neutral, cationic dyes; and ii. one or more
hygroscopic polyols present at less than about 10% v/v of the total
composition.
36. The method of claim 35, wherein the cytological staining
composition comprises water, a water-soluble solvent, a
water-miscible solvent, or mixtures thereof.
37. The method of claim 35, wherein the abnormal cells comprise a
cancer cell.
38. The method of claim 37, wherein the cancer is selected from the
group consisting of cervical, cancers of the reproductive system,
brain, lung, liver, spleen, kidney, renal cell and renal pelvis
cancers, lymph node, small intestine, pancreas, blood cells, bone,
colon/colorectal, stomach, breast, endometrium, prostate, testicle,
ovary, central nervous system, skin, head and neck, thyroid,
para-thyroid, esophagus, bone marrow, hematological cancers,
leukemia, acute promyelocytic leukemia, lymphoma, multiple myeloma,
myelodysplasia, and myeloproliferative disease.
39. The method of claim 37, wherein the cancer cell is a cervical
cancer cell.
40. A composition for cytology staining, comprising: i. Azure C;
ii. one or more hygroscopic polyol(s), said polyol(s) present at
less than about 10% v/v of the total composition; and optionally
iii. water, a water-soluble solvent or water-miscible solvent, or
mixtures thereof.
41. The composition of claim 40, comprising water, a water-soluble
solvent, a water-miscible solvent, or mixtures thereof.
42. The composition of claim 40, wherein Azure C is present in an
amount of about 0.01 to about 0.0001% w/v.
43. The composition of claim 40, wherein said hygroscopic polyol is
selected from the group consisting of glycerol, ethylene glycol,
diethylene glycol, triethylene glycol, propylene glycol,
dipropylene glycol, tripropylene glycol, 1,3-butanediol,
2,3-butanediol, 1,4-butanediol, 3-methyl-1,3-butanediol,
1,5-pentanediol, tetraethylene glycol, 1,6-hexanediol,
2-methyl-2,4-pentanediol, polyethylene glycol, 1,2,4-butanetriol,
1,2,6-hexanetriol, 2-ethyl-2-methyl-1,3-propanediol,
3,3-dimethyl-1,2-butanediol, 2,2-diethyl-1,3-propanediol,
2-methyl-2-propyl-1,3-propanediol, 2,4-dimethyl-2,4-pentanediol,
2,5-dimethyl-2,5-hexanediol, 5-hexene-1,2-diol,
2-ethyl-1,3-hexanediol, 2,2,4-trimethyl-1,3-pentanediol, butylene
glycol, pentaerthyritol, mannitol, sorbitol, di-glycerol,
tri-glycerol, tetra-glycerol, erythritol,
2-hydroxymethyl-2-methyl-1,3-propanediol (trimethylolethane),
2-ethyl-2-(hydroxymethyl)-1,3-propanediol (trimethylolpropane), and
1,2,4-hexanetriol and mixtures thereof.
44. The composition of claim 40, wherein the hygroscopic polyol is
present in an amount of less than 5% v/v.
45. The composition of claim 40, wherein the solvent is a
non-flammable, biocompatible solvent.
46. The composition of claim 40, wherein said solvent is water.
47. A composition for cytology staining, comprising: i. dye
component consisting of Azure C; ii. glycerol, wherein said
glycerol is present in an amount less than about 10% v/v of said
composition; and optionally iii. water.
48. The composition of claim 47, wherein i. Azure C is present in
an amount of about 0.004% w/v; and ii. glycerol is present in an
amount of about 3% v/v of said composition.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 14/765,709, filed on Aug. 4, 2015, which is a U.S. national
phase application under 35 U.S.C. .sctn. 371 of International
Application No. PCT/US2014/014563, filed on Feb. 4, 2014, which
claims the priority of U.S. Provisional Application No. 61/760,889,
filed on Feb. 5, 2013.
FIELD OF THE INVENTION
[0002] The subject matter disclosed herein relates generally to
compositions and methods of preparing cytological samples that
preserve cell morphology to detect abnormal cells.
BACKGROUND
[0003] Cytology is the branch of biology dealing with the study of
the formation, structure, and function of cells. As applied in a
laboratory setting, cytologists, cytotechnologists, and other
medical professionals make medical diagnoses of a patient's
condition based on visual examination of a specimen of the
patient's cells. A typical cytological technique is a "Pap smear"
test, in which cells are scraped from a woman's cervix and analyzed
in order to detect the presence of abnormal cells, a precursor to
the onset of cervical cancer. Cytological techniques are also used
to detect abnormal cells and disease in other parts of the human
body.
[0004] Cytological techniques are widely employed because
collection of cell samples for analysis is generally less invasive
than traditional surgical pathological procedures such as biopsies,
whereby a tissue specimen is excised from the patient using
specialized biopsy needles having spring loaded translatable
stylets, fixed cannulae, and the like. Cell samples may be obtained
from the patient by a variety of techniques including, for example,
by scraping or swabbing an area, or by using a needle to aspirate
body fluids from the chest cavity, bladder, spinal canal, or other
appropriate area. The cell samples are placed in solution and
subsequently collected and transferred to a glass slide for viewing
under magnification. Fixative and staining solutions are typically
applied to the cells on the glass slide, often called a cell smear,
for facilitating examination and for preserving the specimen for
archival purposes.
[0005] Traditional staining solutions can be alcohol-based or
contain significant levels of hazardous materials, such as
formaldehyde. A traditional multicolored stain is desirable for
staining cell smears for certain cytological analyses. It is
advantageous to stain the nucleus and the cytoplasm of the specimen
with different colors, so that the nuclear material and cytoplasmic
material can be readily distinguished either visually or by
automated imaging equipment. In one staining practice, the
cytoplasm is transparent, whereas the nucleus is transparent to
opaque. This staining pattern allows the cytologist to distinguish
cells which are morphologically abnormal indicated, for example, by
nuclear material which is excessively large and/or dark in color.
In addition, cytologists find the variety of colors of the
traditional stains, particularly the Papanicolaou stain, helpful to
reduce eye strain and to aid diagnosis.
[0006] However, traditional stains, including the Papanicolaou
stain, are difficult for an automated system to analyze. The
variety of colors in the cytoplasm from traditional stains, which
are straightforward for the human eye to distinguish, are not
readily analyzed with automated imaging systems because they
contrast to varying degrees with the traditional blue hematoxylin
stain of the nucleus. The varying contrast makes automated analysis
unreliable. Many diagnostic assays depend on the evaluation of
cytological and histological components. One requirement of these
evaluations is the adhesion of these components to a solid
substrate, such as a glass or plastic microscope slide. Once
immobilized by adhesion, these components may be processed further
to gain diagnostic information.
[0007] Several cytological/histological adhesion reagents have been
developed. Although effective, adhesive reagents to some degree
lose their adhesive properties and do not remain stable once
coated, applied, and dried on a solid substrate. This results in
the need to prepare freshly coated slides for optimal routine
use.
[0008] What is therefore needed is a cytological staining
composition that provides a high contrast between the nucleus and
the cytoplasm of a cell but does not require freshly coated slides
for optimal performance. It is evident that there is an unmet need
for cytological staining compositions that would be considered
non-hazardous material, are water-soluble and can be used in a
one-step cytological preparation protocol.
BRIEF SUMMARY
[0009] In one aspect, the present subject matter is directed to a
composition for staining cytological material comprising: i. a dye
component consisting essentially of one or more water-soluble, Ph
neutral, cationic dyes, ii. A hygroscopic polyol that is present in
an amount less than about 10% v/v of the total composition, and
optionally iii. A water-soluble solvent, water or a water-miscible
solvent.
[0010] In one aspect, the present subject matter is directed to a
composition for staining cytological material comprising: i. a dye
component consisting essentially of one or more water-soluble, Ph
neutral, anionic dyes, ii. A hygroscopic polyol that is present in
an amount less than about 10% v/v of the total composition, and
optionally iii. A water-soluble solvent, water or a water-miscible
solvent.
[0011] In another aspect, the present subject matter is directed to
a method of preparing a cytological preparation comprising
contacting a cell sample with a composition comprising: i. a dye
component consisting essentially of one or more water-soluble, Ph
neutral, cationic dyes, ii. A hygroscopic polyol that is present in
an amount less than about 10% v/v of the total composition, and
optionally iii. A water-soluble solvent, water or a water-miscible
solvent.
[0012] In another aspect, the present subject matter is directed to
a method of analyzing a cell sample comprising, a) contacting the
cell sample with a composition comprising: i. a dye component
consisting essentially of one or more water-soluble, Ph neutral,
cationic dyes; ii. A hygroscopic polyol that is present in an
amount less than about 10% v/v of the total composition, and
optionally iii. A water-soluble solvent, water or a water-miscible
solvent; and b) subjecting the sample to an analysis or
evaluation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The present invention will be better understood and
features, aspects and advantages other than those set forth above
will become apparent when consideration is given to the following
detailed description thereof. Such detailed description makes
reference to the following drawings, wherein:
[0014] FIG. 1 is a flow chart for a one-step process of staining a
cytological sample using a cytological staining composition
disclosed herein and a substrate having a polycationic charge
coating.
[0015] FIG. 2 is a flow chart for a process of staining a
cytological sample using a cytological staining composition
disclosed herein and a substrate having a polycationic charge
coating.
[0016] FIG. 3 depicts abnormal cervical cells that were stained
with the staining solution as described herein and subsequently
immobilized onto a microscope slide having a polycationic charge
coating. While the entirety of the cells and cell groups are
readily visualized, the detail of the nucleus is extremely sharp in
definition and can easily be distinguished for size, shape, and
texture.
[0017] FIG. 4 depicts the same cells on the same slide in FIG. 3
that were originally stained with the staining solution. The sample
can be subsequently restained with standard Papanicolaou stain.
Note that the nuclear detail is less defined and more difficult to
ascertain.
[0018] FIG. 5 depicts abnormal cervical cells that were stained
with the staining solution as described herein and subsequently
immobilized onto a microscope slide having a polycationic charge
coating. While the entirety of the cells and cell groups are
readily visualized, the detail of the nucleus is extremely sharp in
definition and can easily be distinguished for size, shape, and
texture.
[0019] FIG. 6 depicts the same cells on the same slide in FIG. 3
that were originally stained with the staining solution and
subsequently restained with standard Papanicolaou stain. Note that
the nuclear detail is less defined and more difficult to
ascertain.
[0020] FIG. 7 depicts abnormal cervical cells that were stained
with the staining solution as described herein and subsequently
immobilized onto a microscope slide having a polycationic charge
coating. While the entirety of the cells and cell groups are
readily visualized, the detail of the nucleus is extremely sharp in
definition and can easily be distinguished for size, shape, and
texture.
[0021] FIG. 8 depicts the same cells on the same slide in FIG. 3
that were originally stained with the staining solution and
subsequently restained with standard Papanicolaou stain. Note that
the nuclear detail is less defined and more difficult to
ascertain.
DETAILED DESCRIPTION
[0022] The subject matter disclosed herein is directed to
compositions for staining cytological material and the methods of
their use. The compositions provide excellent contrast of the
cellular components including the nucleus and related structures
relative to the cytoplasm and its related structures. Additional
advantageous aspects of the present invention are that the
compositions are pH neutral, i.e., not corrosive, are
aqueous-based, i.e., not flammable and can be used in a one-step
system for characterizing cytological material. Because the
compositions can provide superior contrast, they are suitable for
use in a convenient one-step system for characterizing cytological
material. In this aspect, the compositions do not require equipment
that is complicated, expensive and often inaccessible. Too often,
the need for such equipment results in the foregoing of cytological
screenings. Consequently, screenings are not performed and an
opportunity for early diagnosis is missed. Accordingly, the
compositions described herein not only can offer superior contrast
but also advantageously provide almost unlimited access to cytology
screening.
[0023] Advantageously, the present compositions do not require the
use of a standard cover slide as is required in known staining
fixatives. The compositions are able to retain cell morphology for
a period of time such that a standard cover slide is not required.
Further, the compositions do not contain hazardous levels of
organic solvents or other components that are not biocompatible.
Preferably, the compositions consist essentially of Azure C,
glycerol and optionally water. In another aspect, the present
subject matter is directed to a method of characterizing a cell
sample comprising contacting the cell sample with a composition
described herein and subjecting the sample to analysis to determine
the presence or absence of abnormal cells.
[0024] As used herein, the term "water-soluble, pH neutral,
cationic dye" is intended to mean a compound or biologic that is a
dye or stain useful for staining cells or cell components whereby
the dye or stain has the following properties: The dye or stain is
preferably readily soluble at useful concentrations in water at
20.degree. C. and at the final pH of the composition; the dye or
stain is pH neutral, which means that when dissolved in an aqueous
solution, the dye or stain does not alter the pH of the solution to
a significant degree. In other words, the final pH of the solution
is from about 6-8; and the dye or stain is cationic, which means
that the dye or stain is capable of ionizing to a cationic ion in
an aqueous solution. Preferably, the stain used in this process is
from a class of "basic ionized" stains and contains positively
charged functional groups. These charges do not compete for the
negative binding sites on the positively charged slide surface.
Therefore, cells stained with these types of stains bind readily
and strongly adhere to the positively charged slides. As these
stains are aqueous based and are used at a relatively neutral pH of
6.0-8.0, they would not be considered corrosive. They are also
non-flammable.
[0025] As used herein, the term "water-soluble, pH neutral, anionic
dye" is intended to mean a compound or biologic that is a dye or
stain useful for staining cells or cell components whereby the dye
or stain has the following properties: The dye or stain is
preferably readily soluble (e.g., 50 g/L) in water at 20.degree. C.
and at the final pH of the composition; the dye or stain is pH
neutral, which means that when dissolved in an aqueous solution,
the dye or stain does not alter the pH of the solution to a
significant degree. In other words, the final pH of the solution is
from about 6-8; and the dye or stain is anionic, which means that
the dye or stain is capable of ionizing to an anionic ion in an
aqueous solution.
[0026] As used herein, the term "hygroscopic" is intended to mean a
property of a polyol to attract and hold water molecules from the
surrounding environment. This can be achieved by absorption or
adsorption. The polyol may physically change as it absorbs or
adsorbs water, for example, by an increase in its volume,
stickiness or ability to hydrate substances that the polyol is
contacting.
[0027] As used herein, the term "polyol" is intended to encompass
aliphatic diols and triols and derivatives thereof. Preferably, the
polyol is a liquid.
[0028] The term "biocompatible" refers to solvents that do not
induce excessive toxic or excessive unwanted effects on the cell
samples when employed in useful amounts. These effects can include
dehydration and destabilization of cytological architecture.
[0029] The term "organic solvent" refers to an organic compound
with solvent properties or a mixture of organic compounds with
solvent properties. Generally, in the field of the invention,
organic solvents belong to various chemical classes such as
hydrocarbons, ketones, alcohols, carboxylic acid esters, and the
like. Preferably, the cytological staining solutions described
herein are water-based and contain low, trace or zero amounts of
organic solvents.
[0030] The term "water-soluble solvent" means a liquid that
solvates the components of the composition, is substantially inert
and will dissolve in water. This is intended to mean that the
solvent does not affect pH or any other property of the composition
and the solvent does not itself contain a dye prior to solvating
the composition. A water-soluble solvent may also be an organic
solvent. However, as disclosed herein, the final composition will
contain a solvent system made up of water, water-soluble solvent(s)
and/or water-miscible solvent(s) such that the composition is,
unlike known compositions, biocompatible and non-flammable.
[0031] The term "miscible" refers to the ability of a liquid to mix
evenly into another liquid.
[0032] As used herein, the term "hazardous material" or "hazardous
level" refers to known regulatory standards for either use or
exposure in humans; hazardous materials transport regulations
and/or regulations for incineration or disposal.
[0033] As used herein, the term "non-flammable" is intended to mean
a composition or solvent which is never ignited by an igniting
source irrespective of its mixing ratio with air if its temperature
is in a practical temperature range. In the above-described
definitions, the practical temperature range is from -40.degree. C.
to +100.degree. C. On the other hand, the term "inflammable" as
used herein is intended to mean the composition or solvent which,
upon touching an igniting source, is ignited if its temperature is
in a practical temperature range, and its mixing ratio with air is
a predetermined value.
[0034] The term "abnormal cell" refers to any diseased or
pathological cell. Abnormal cells demonstrate abnormal
morphological characteristics when viewed microscopically. These
abnormal morphological characteristics can be observed by employing
the cytological staining compositions disclosed herein and simple
light microscopy. Non-limiting examples of abnormal cells include
pre-cancerous changes as well as true cancerous cells.
[0035] The subject matter described herein is a convenient process
for the staining and temporary, i.e., hours, days and weeks,
preservation of stained cytological samples such that the samples
may be evaluated for risk stratification and subsequently processed
with alternate staining or other processes. This process utilizes a
water soluble stain reagent and a water soluble solvent, which may
be an organic component, to preserve cytological morphology and
architecture. Optimally, the process can incorporate pre-coated
substrates. Additionally, the methods can further comprise
contacting the cells that have been stained with a composition
described herein with a subsequent staining solution, such as
standard Papanicolaou stain. This overstaining process does not
require de-staining steps prior to the use of the subsequent
staining solution.
[0036] In an embodiment, the present subject matter is directed to
a composition for cytology staining, comprising: i. a dye component
consisting essentially of one or more water-soluble, pH neutral,
cationic dyes; ii. a hygroscopic polyol; and optionally iii. a
water-soluble solvent. In this embodiment, the dye component will
not contain an anionic dye or a mixture of dyes that will interfere
with the immobilization of the cell sample or delineation of
cellular architecture.
[0037] Useful cationic dyes include crystal violet, acriflavine,
bismarck brown, malachite green, methyl green, Victoria pure blue
BO, and Azure C, and analogues thereof. Examples of cationic dyes
include those listed in Table 1:
TABLE-US-00001 TABLE 1 Cationic dyes Basic blue 8 42563
Triarylmethane Victoria blue 4R Basic blue 9 52015 Thiazin
Methylene blue Basic blue 12 51180 Oxazin Nile blue A Basic blue 15
44085 Triarylmethane Night blue Basic blue 17 52040 Thiazin
Toluidine blue O Basic blue 20 42585 Triarylmethane Methyl green
Basic blue 26 44045 Triarylmethane Victoria blue B Basic brown 1
21000 Azo Bismarck brown Y Basic fuchsin -- Triarylmethane Basic
fuchsin Basic green 4 42000 Triarylmethane Malachite green Basic
green 5 52020 Thiazine Methylene green Basic orange 14 46005
Acridine Acridine orange Basic red 2 50240 Safranin Safranin O
Basic red 5 50040 Eurhodin Neutral red Basic red 9 42500
Triarylmethane Pararosanilin Basic violet 2 42520 Triarylmethane
New Fuchsin Basic violet 3 42555 Triarylmethane Crystal violet
Basic violet 4 42600 Triarylmethane Ethyl violet Basic violet 10
45170 Rhodamine Rhodamine B Basic violet 14 42510 Triarylmethane
Rosanilin Basic yellow 1 49005 Thiazole Thioflavine T Basic yellow
2 41000 Diarylmethane Auramine O
[0038] A particularly useful dye is Azure C. Azure C has the
following chemical structure:
##STR00001##
[0039] In useful embodiments, the dye component consists
essentially of a cationic dye. In these embodiments, the
composition does not contain an anionic dye. In a preferred
embodiment, the dye component consists of a single dye, such as
Azure C. This means that the composition for cytology staining
contains a single dye, though the composition can comprise other
components and adjuvants that are not dyes.
[0040] Cationic dyes are preferred. In a preferred method employing
the cytological staining compositions described herein, the
cytological sample is associated with a substrate. Preferably, this
substrate has a cationic coating. Such a coating provides a charge
potential that binds biological samples of interest to the
substrate. For example, a microscopic slide can have a coating that
facilitates fixation of the sample to the slide. FIGS. 1 and 2
depict exemplary processes and FIGS. 3-8 depict cell staining on
such substrates. An exemplary substrate of this kind is described
in PCT/US2005/033938 (WO/2006/034385), herein incorporated by
reference in its entirety. Protocols that employ poly-L lysine,
poly-D lysine and silanol substrates are also contemplated.
[0041] Preferably, the cytological staining compositions described
herein can be employed in a one-step cytological sample preparation
protocol. In customary cytological staining solutions, the sample
must first be immobilized on a substrate. Only then is it practical
to stain the immobilized sample. In contrast, the cytological
staining compositions described herein can advantageously be used
in a solution that immobilizes the sample and concurrently stains
the nuclear and/or cytoplasmic structures of the cells.
[0042] Anionic dyes can also be used in the cytological staining
compositions described herein. However, because of the convenience
and oftentimes necessity for a one-step, straightforward
cytological preparation, the cationic dyes disclosed herein are
preferred. Useful anionic dyes include those listed in Table 2 as
well as homologues thereof:
TABLE-US-00002 TABLE 2 Anionic dyes Acid black 1 20470 Azo Amido
black 10B Acid blue 22 42755 Triarylmethane Water blue Acid blue 93
42780 Triarylmethane Methyl blue Acid fuchsin 42685 Triarylmethane
Acid fuchsin Acid green 42095 Triarylmethane Light green SF
yellowish Acid green 1 10020 Nitroso Naphthol green B Acid green 5
42095 Triarylmethane Light green SF yellowish Acid magenta 42685
Triarylmethane Acid fuschsin Acid orange 10 16230 Azo Orange G Acid
red 4 14710 Azo Azo-eosin Acid red 26 16150 Azo Xylidine ponceau
Acid red 29 16570 Azo Chromotrope 2R Acid red 44 16250 Azo Ponceau
6R Acid red 51 45430 Fluorone Erythrosin B Acid red 52 45100
Xanthene Lissamine rhodamine B Acid red 66 26905 Azo Biebrich
scarlet Acid red 73 27290 Azo Woodstain scarlet Acid red 87 45380
Fluorone Eosin Y ws Acid red 91 45400 Fluorone Eosin B Acid red 92
45410 Fluorone Phloxine B Acid red 94 45440 Fluorone Rose bengal
Acid red 101 50085 Quinone-Imine Azocarmine G Acid red 103 50090
Quinone-Imine Azocarmine B Acid roseine 42685 Triarylmethane Acid
fuchsin Acid rubin 42685 Triarylmethane Acid fuchsin Acid violet 19
42685 Triarylmethane Acid fuchsin Acid yellow 1 10316 Nitro
Naphthol yellow S Acid yellow 7 56205 Ketonamine Lissamine flavine
FF Acid yellow 9 13015 Nitro Fast yellow Acid yellow 23 19140 Azo
Tartrazine Acid yellow 24 10315 Nitro Martius yellow Acid yellow 36
13065 Azo Metanil yellow Acid yellow 73 45350 Fluorone Fluorescein
Acid yellow 85 -- Azo Coomassie fast Yellow G Acid yellow S 10316
Nitro Naphthol yellow S Acid yellow T 19140 Azo Tartrazine
[0043] Further, if an anionic dye is used, then a cationic dye is
excluded and vice versa.
[0044] Useful amounts of dye in the compositions are from about
0.01 to about 0.0001 w/v of the total composition. Preferably, the
amount is from about 0.009 to about 0.001% w/v. More preferably,
the amount is from about 0.005 to about 0.003% w/v. Most
preferably, the amount is about 0.004% w/v.
[0045] Staining the target cells with the staining composition
disclosed herein provides excellent morphological detail for
identification of cells in a sample, for example abnormal cells.
Other critical information useful for a more thorough diagnosis,
such as the presence or absence of specific protein markers within
the cell can also be obtained using the methods described herein.
To ascertain such protein marker details from these abnormal cells,
the presently described methods can incorporate known
immunochemistry techniques. These techniques involve the
application of specific antibodies designed to detect the protein
markers of interest, along with some type of signaling entity to
indicate that the antibody has indeed attached to the proteins of
interest in the cells. These signaling entities can include, but
are not limited to, conjugated dyes, fluoro chromes,
chemilluminescent reactants, and radioactive isotopes. In addition
to providing rapid morphological detail of cytological samples, the
compositions and methods disclosed herein allow for the target
cells to be subsequently stained using such immuno-chemistry
techniques as described above.
[0046] Useful hygroscopic polyols include glycerol, ethylene
glycol, diethylene glycol, triethylene glycol, propylene glycol,
dipropylene glycol, tripropylene glycol, 1,3-butanediol,
2,3-butanediol, 1,4-butanediol, 3-methyl-1,3-butanediol,
1,5-pentanediol, tetraethylene glycol, 1,6-hexanediol,
2-methyl-2,4-pentanediol, polyethylene glycol, 1,2,4-butanetriol,
1,2,6-hexanetriol, 2-ethyl-2-methyl-1,3-propanediol,
3,3-dimethyl-1,2-butanediol, 2,2-diethyl-1,3-propanediol,
2-methyl-2-propyl-1,3-propanediol, 2,4-dimethyl-2,4-pentanediol,
2,5-dimethyl-2,5-hexanediol, 5-hexene-1,2-diol,
2-ethyl-1,3-hexanediol or 2,2,4-trimethyl-1,3-pentanediol, butylene
glycol, pentaerthyritol, mannitol, sorbitol, di-glycerol,
tri-glycerol, tetra-glycerol, erythritol,
2-hydroxymethyl-2-methyl-1,3-propanediol (trimethylolethane),
2-ethyl-2-(hydroxymethyl)-1,3-propanediol (trimethylolpropane),
1,2,4-hexanetriol and mixtures thereof. Preferably, the glycol is
glycerol or sorbitol. Most preferably, at least one of the
polyol(s) contained in the composition is glycerol. Most
preferably, the only polyol contained in the composition is
glycerol.
[0047] Useful amounts of polyol are less than about 10% w/w of the
total composition, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% and
1% or less. Preferably, the amount of polyol present is less than
about 5% w/v. Preferred compositions include polyol from about
0.001% to about 1.0% v/v. Most preferably, the amount of polyol
present is about 0.05% w/v.
[0048] Useful solvents include water, water-soluble solvents and
water-miscible solvents. Non-flammable solvents are those that can
be used in concentrations that are below the flash point in the
final composition. These include water and preferably simple
alcohols. Most preferably, the solvent is water.
[0049] There exists a need for more efficient cytological sample
preparation systems for improving the consistency, repeatability,
and accuracy of sample preparation and analysis. In addition,
manual intervention and/or manual processing in the sample
preparation process may increase the likelihood that a sample will
become contaminated. Any further analysis of such a contaminated
sample may lead to an improper medical diagnosis. Manual
intervention/manual processing may also increase the risk that the
operator can become infected with any sample-borne virus or disease
that may be present in the specimen. There exists a further need
for more efficient cytological sample preparation systems that
eliminate, reduce, or otherwise minimize, where possible, manual
intervention and/or manual processing during sample preparation,
such as, for example, through automation. Such a cytological sample
preparation system should desirably be, for example, relatively
easy to use and adaptable for processing a varying numbers of
samples. The compositions and methods disclosed herein meet these
needs.
[0050] In an embodiment, the subject matter disclosed herein is
directed to methods of preparing a cytological preparation
comprising: a) contacting a cell sample with a composition
comprising: i. a dye component consisting essentially of one or
more water-soluble, pH neutral, cationic dyes; ii. a hygroscopic
polyol; and optionally iii. a water-soluble solvent. In this
embodiment, the method can further comprise simultaneously
contacting and immobilizing the cell sample onto a pre-coated
substrate having a cationic coating as described elsewhere
herein.
[0051] In an embodiment, the subject matter disclosed herein is
directed to methods of preparing a cyto logical preparation
comprising: a) contacting a cell sample with a composition
comprising: i. a dye component consisting essentially of one or
more water-soluble, pH neutral, anionic dyes; ii. a hygroscopic
polyol; and optionally iii. a water-soluble solvent. In this
embodiment, the cell sample has already been immobilized on a
pre-coated substrate having a cationic coating as described
elsewhere herein.
[0052] The cell sample is as described elsewhere herein. In useful
embodiments, the cell sample is susceptible to containing abnormal
cells of a subject.
[0053] In embodiments, the abnormal cell is a cancerous,
pre-cancerous or atypical cell. Cells of the following types of
cancers are of particular interest: a solid tumor, such as cervical
or cancer of the reproductive system, brain, lung, liver, spleen,
kidney (such as renal cell and renal pelvis), lymph node, small
intestine, pancreas, blood cells, bone, colon/colorectal, stomach,
breast, endometrium, prostate, testicle, ovary, central nervous
system, skin, head and neck, esophagus, or bone marrow, or a
hematological cancer, such as leukemia, acute promyelocytic
leukemia, lymphoma, multiple myeloma, myelodysplasia,
myeloproliferative disease, or refractory anemia. In particular,
the cancer cell is cervical or an atypical cell associated with
cervical cancers or disorders.
[0054] In embodiments, the cell sample is associated with a
substrate. Useful substrates are selected from the group consisting
of a microscope slide, a cuvette, a coverslip and a microtiter
tray. Preferably, the substrate is a microscopic slide. In a
preferred embodiment, the method does not comprise associating a
standard cover slide with the sample that is associated with the
substrate. A standard cover slip is a plastic, glass or other
material that is transparent and can be hard or flexible. A
standard cover slip does not include a liquid cover slip.
[0055] In an embodiment, the subject matter described herein is
directed to a method of characterizing a cytological sample
comprising: a) contacting a cell sample with a composition
containing a cationic dye as disclosed herein, wherein the sample
is simultaneously associated with a substrate, and b. subjecting
said sample to an assay to determine the presence or absence of
abnormal cells. In other embodiments, the subject matter described
herein is directed to a method of characterizing a cytological
sample comprising: a) contacting a cell sample with a composition
disclosed herein, and b. subjecting said sample to an assay to
determine the presence or absence of abnormal cells.
[0056] In these embodiments, the assay is any analyses or
evaluation employed to determine the presence or absence of
abnormal cells. These analyses are known in the art and can be
automated or can be performed by a human such as a technician,
cytologist or pathologist.
[0057] In an embodiment, the subject matter described herein is
directed to a method of preparing a cyto logical sample
comprising:
[0058] a. Depositing cells onto a microscope slide, wherein said
slide has a polycationic coating;
[0059] b. Rinsing excess cells from the slide by contacting with
Composition Stain;
[0060] c. Allowing the Composition Stain to contact the slide for
about 5 seconds, 30 seconds, 1 minute or longer;
[0061] d. Removing excess Composition Stain from the slide; and
[0062] e. Optionally, contacting the sample on the slide with a
liquid cover slip or additional composition stain (about 25
.mu.l).
[0063] The cytological preparation is ready for cytological
evaluation. If additional staining or alternate processing is
required after the initial evaluation of the processed slide, the
technician simply has to rinse the slide in Deionized Water and
proceed directly to the subsequent process. Cellular morphology and
architecture of the sample will have been preserved.
[0064] In an embodiment, the subject matter described herein is
directed to a method of preparing a cytological sample
comprising:
[0065] a. Utilizing Composition Stain to wash and concentrate
cells;
[0066] b. Depositing stained cells in Composition Stain onto a
microscope slide, wherein said slide has a polycationic
coating;
[0067] c. Allowing cells to associate with the slide (typically 1
minute to 10 minutes);
[0068] d. Remove excess Composition Stain from the slide; and
[0069] e. Optionally, adding a Liquid Cover Slip or additional
composition stain to slide (about 25 .mu.l) and allowing it to
spread over slide.
[0070] The cytological preparation is ready for cytological
evaluation. If additional staining or alternate processing is
required after the initial evaluation of the processed slide, the
technician simply has to rinse the slide in Deionized Water and
proceed directly to the subsequent process. Cellular morphology and
architecture of the sample will have been preserved.
[0071] The cytological staining compositions described herein are
able to prevent, mitigate or delay collapse of the cytological
architecture of a cytological preparation. Cytological preparation
refers to any sample containing biological materials, but in
particular, refers to a sample containing cells. A disadvantage of
known stains is that while they are capable of staining cells to
varying degrees, they can contain high levels of alcohols or other
solvents and components that facilitate collapse or degradation of
cell morphology. While not being bound to any theory, it is
believed that when the cell dehydrates, the structures within the
cell compress and collapse. The cytological staining compositions
described herein can stain the nucleus and cytoplasm with
exceptional delineation of cell architecture and, unlike known
compositions, they additionally prevent, mitigate or delay cell
compression and collapse. As a result, components of the nucleus
and cytoplasm of cells in a sample are prepared with excellent
delineation. Additionally, it is not necessary to place a cover
slip on the sample to help preserve or affix the sample.
[0072] In embodiments, the sample can be contacted with water to
rehydrate any contents therein that have become dehydrated.
[0073] The figures disclosed elsewhere herein show the delineation
of cellular architecture provided by the cytological staining
compositions described herein. In addition to the benefits of being
economical and not considered to be a hazardous material, the
presently disclosed staining solutions perform better than the gold
standard PAP stains.
[0074] While staining solutions are known, they are alcohol-based
or contain hazardous components. The alcohol-based compositions
contain much higher levels of alcohol than the presently described
compositions. In all embodiments, the cytological staining
compositions described herein cannot be described correctly as
alcohol-based since the level of polyol(s) is less than 10%.
Preferably, the remaining solvent component of the composition is
made up of mostly or entirely of water. This provides the benefit
of a non-flammable composition. Accordingly, the cytological
staining compositions described herein are safer to handle and
transport than traditional stains. Additionally, known compositions
contain levels of fixatives such as formaldehyde. While
formaldehyde is capable of fixing cells, it is also not
biocompatible at useful levels. Often, solutions containing such
levels of chemicals are classified as hazardous. This makes
providing the staining solutions unnecessarily regulated and
expensive in view of the cytological staining solutions disclosed
herein. The cytological staining compositions disclosed herein
overcome these problems.
[0075] Preferably, the methods described herein utilize a
pre-coated substrate. The pre-coated substrate has a net positive
(cationic) coating. This coating facilitates association of the
sample to the substrate. When the composition contains only a
cationic dye(s), the composition will not interfere with the
function of the coating. Advantageously, the composition can be
used concurrently during immobilization of the sample to the
substrate.
[0076] Cells have a net negative charge on their outer surface.
Glass slides used to immobilize cells for subsequent processing
have been treated to provide a new positive charge on their
surface. This charge difference between the cells and the treated
slides creates the electrostatic adhesion to immobilize the cells
on the slide surface. Most cytology staining techniques rely on
"acid ionized" stains that contain negatively charged functional
groups. As such, these stains can compete with the negative charged
cell surfaces for binding sites on the positively charged glass
slides. This results in very poor adhesion of cells to the slide
surface when they have been stained in this manner. Many of these
stains are also alcohol based and considered a hazardous material
due to the corrosive qualities of the low pH (acid) and the
flammability due to the high alcohol content.
[0077] The methods described herein are combinable with aspects of
known cytology preparation processes to simplify the entire sample
preparation process, save considerable time, and substantially
increase processing throughput.
[0078] Additionally, FIGS. 3, 5 and 7 show excellent nuclear
details using a straightforward method with the stain solution as
disclosed herein. The level of detail is optimal for analysis.
While automated systems employing most other stains such as
standard Papanicolaou stain require algorithms to attempt with some
success to filter out chromagens, the use of the presently
disclosed methods and stain solution provide a sample that is ready
for immediate analysis. Thus, the use of the present stain
solutions is not only simpler than art methods, the results provide
superior morphology and detail as well as unmatched access to
screening where the equipment needed in standard screening is not
available.
[0079] Moreover, FIGS. 4, 6 and 8 show that the same sample can be
overstained with a traditional stain should the use of a
traditional stain be desired. This overstaining can be accomplished
without first destaining the sample. Though surprisingly the
presently disclosed stain solutions and methods provide exceptional
cellular morphology and details, an advantage of these methods and
stain solutions is that subsequent overstaining of the very same
sample can be performed if desired. Overstaining of the very same
sample entirely eliminates the high probability of not obtaining
more of the same abnormal cells when analyzing a different sample
from the same subject. The different sample may not contain
abnormal cells at all since the abnormality may be rare, at an
early stage or simply not observed among all the normal cells in
the sample. Indeed, detecting abnormal cells early requires that
relatively few abnormal cells present are observed. Accordingly,
overstaining the same cell sample that has an observed abnormality
eliminates the possibility that these abnormal cells detected
initially are subsequently not observed in a sample that is
subsequent or different from the original sample taken from the
same subject.
[0080] Once the stained sample has been prepared, a small amount
(20-50 .mu.l) of a simple and benign solution is added to the cell
deposition. This solution contains a percentage of glycerol in
Deionized water and is also at a relatively neutral pH of 4.0-6.0.
Though not tied to a particular theory, the hygroscopic nature of
glycerol helps retain water within the cells which prevents
morphology degradation and other drying artifacts which could
negatively impact the evaluation. Surprisingly, the cell morphology
is not affected by the cytological staining compositions disclosed
herein, which have been shown to provide excellent delineation of
cellular structure and retention of cellular morphology. By
providing this osmotic and hydrostatic pressure balance, not only
are the individual cells preserved but also the tertiary
cytological architecture is maintained in such complex components
as glandular cells or clusters.
[0081] Current cytological staining and evaluation processes
utilize the following steps before the sample can be reviewed:
Adhere the sample to a glass slide or other substrate; Stain the
sample, often with alcohol based stains; Remove excess stains from
the slide with alcohol; Remove residual water and alcohols with a
clearing agent, such as xylene; Applying a mounting media and
cover-slip to the slide and allowing it to dry. While effective,
the art processes require that the sample first be adhered to a
glass slide prior to staining. Also, the majority of cytological
staining procedures require multiple stains of separate aqueous and
organic base solvents. This requires special handling and requires
multiple containers and rinse steps to transition between the
different stains. If additional staining or alternate processing is
required after the initial evaluation of the processed slide, the
technician would either need to prepare another sample or remove
the cover-slip and try and reprocess the original slide sample.
[0082] In summary, the art processes can include these steps:
[0083] Sample is pre-processed to remove unwanted materials and
concentrate the cells. [0084] (i.e.: Centrifugation, Filter
Adhesion, etc. . . . ) [0085] Cells are deposited onto a charged
microscope slide [0086] Excess cells are rinsed from slide [0087]
Slide may be stored in an Alcohol solution until ready for
staining. [0088] Slide is rinsed with Deionized Water to remove
residual Alcohol [0089] Slide is exposed to a nuclear stain [0090]
Aqueous based such as Hematoxylin [0091] Slide is rinsed with
Deionized Water to remove residual nuclear stain [0092] Slide is
rinsed with Alcohol to remove residual Deionized Water [0093] Slide
is exposed to a cytoplasmic stain [0094] Alcohol based such as
EA/OG [0095] Slide is rinsed with Alcohol to remove residual
cytoplasmic stain [0096] Slide is rinsed with Clearing Agent to
remove residual Alcohol [0097] Clearing agent is usually Xylene or
Xylene substitute [0098] Mounting Media is added to slide [0099]
Cover-Slip is mounted to slide and Mounting Media [0100] Mounting
Media is allowed to dry adequately such that the Cover-Slip does
not move [0101] Slide is ready for cytological evaluation. As noted
above, if additional staining or alternate processing is required
after the initial evaluation of the processed slide, the technician
would either need to prepare another sample or remove the
cover-slip and try and reprocess the original slide sample.
[0102] By preparing a subsequent sample, there is a chance that
"rare event" cells may not be included in the new slide. Also, the
arrangement of the cells on the new slide will be totally different
from the original, thereby negating the ability to look at the very
same cells of interest using X-Y coordinate location. If the
cover-slip has to be removed from the original sample for
reprocessing, it is possible that the sample material may be
damaged or some of the critical "rare event" cells may be also
removed. Even if morphology and "rare-event" cells are maintained,
this is an arduous and time consuming task.
[0103] The methods described herein eliminates many if not all
these issues while providing the following: Use of a single aqueous
based stain to provide excellent nuclear detail and cytoplasm
staining; Simultaneously staining of the sample while processing
(One step sample preparation and staining); Adhesion of the stained
sample cells onto a pre-coated slide (if employed); Temporary
preservation of the sample morphology and staining without the need
for a cover-slip to allow initial evaluation of the sample for
"risk stratification" to determine if additional processing is
required; Immediate transition to subsequent processing, such as
the standard Papanicolaou stain. This process and component design
could significantly reduce the initial staining and evaluation
complexity currently required for cytological evaluations while
providing rapid transition to alternate processing protocols from
the same slide.
[0104] Procurement of the sample material from the subject for the
cytological evaluation utilizes any methods known in the art. In
conjunction with sample collection, samples may be exposed to other
agents such as buffers, diluents, extraction or chromatographic
media, cross-linking agents, blocking agents, denaturing agents,
etc., to stabilize or otherwise prepare the sample for processing
within a desired assay.
[0105] Once the sample has been prepared as described herein, any
known method of analyses can be employed. Such methods include
visual assessment with or without the aid of a magnifying device
such as a microscope. A system for imaging a cytological sample
including nuclear material and cytoplasmic material that includes
an optical instrument and one or more light sources can be
employed. Other equipment and techniques are known to those of
skill in this field such as cytologists, technicians and
pathologists, and can be used in combination with the compositions
and methods disclosed herein.
[0106] In addition to the cytological staining compositions and
methods of their use, the subject matter disclosed herein is
directed to kits comprising the compositions, substrates,
components, accessories, reagents and other related materials for
practicing the sample preparation methods. These kits are
clinically useful for preparing the sample to identify the presence
or absence of an abnormal cell. The kits include an amount of
cytological staining solution and may also include a polycationic
coated substrate. The foregoing kit components are generally
assembled in a collective packaging unit, which may include written
or otherwise user-accessible instructions detailing the sample
collection, handling and/or processing methods of the
invention.
[0107] Kits for practicing the cytological preparation methods can
include a suitable container or other device for collecting,
storing, handling and/or processing a biological sample. A range of
suitable collection devices is contemplated. For example, simple
sterile containers or reservoirs are provided. A variety of solid
phase devices, including microscopic glass slides, membranes,
filters and like media, are provided to receive or partition
selected liquid or solid fractions of the sample, or to receive or
partition cells or cellular constituents from the sample. A wide
variety of such sample collection devices are widely known or
described in the literature, which can be readily adapted for use
within specific embodiments.
[0108] The following examples are offered by way of illustration
and not by way of limitation.
Examples
[0109] 1. Preparation of Cytological Sample
[0110] Sample is pre-processed to remove unwanted materials and
concentrate the cells (i.e.: Centrifugation, Filter Adhesion,
etc.);
[0111] Cells are deposited onto a charged microscope slide;
[0112] Use Composition Stain to rinse excess cells from slide;
[0113] Incubate Composition Stain on slide (about 60 seconds);
[0114] Remove excess Composition Stain from slide;
[0115] Add a Liquid Cover Slip to slide (about 25 .mu.l) and
allowing it to spread over slide;
[0116] Slide is ready for cytological evaluation.
[0117] If additional staining or alternate processing is required
after the initial evaluation of the processed slide, the technician
simply has to rinse the slide in Deionized Water and proceed
directly to the subsequent process. Cellular morphology and
architecture of the sample will have been preserved.
[0118] 2. Preparation of Cytological Sample in a One Step Process
Use Composition Stain to Wash and Concentrate Cells;
[0119] Stained cells in Composition Stain are deposited onto a
charged microscope slide;
[0120] Allow cells to settle on slide for about 4 minutes;
[0121] Remove excess Concept Stain from slide;
[0122] Add Concept Liquid Cover Slip or additional composition
stain to slide (about 25 .mu.l) and allowing it to spread over
slide;
[0123] Slide is ready for cytological evaluation.
[0124] If additional staining or alternate processing is required
after the initial evaluation of the processed slide, the technician
simply has to rinse the slide in Deionized Water and proceed
directly to the subsequent process. Cellular morphology and
architecture of the sample will have been preserved.
[0125] 3. Delineation of Cellular Architecture
[0126] FIG. 3 depicts abnormal cervical cells that were stained
with the staining solution as described herein and subsequently
immobilized onto a microscope slide having a polycationic charge
coating. While the entirety of the cells and cell groups are
readily visualized, the detail of the nucleus is extremely sharp in
definition and can easily be distinguished for size, shape, and
texture. FIG. 4 depicts the same cells on the same slide in FIG. 3
that were originally stained with the staining solution and
subsequently restained with standard Papanicolaou stain. Note that
while the cellular features are preserved, the nuclear detail is
less defined and more difficult to ascertain. FIG. 5 depicts
abnormal cervical cells that were stained with the staining
solution as described herein and subsequently immobilized onto a
microscope slide having a polycationic charge coating. While the
entirety of the cells and cell groups are readily visualized, the
detail of the nucleus is extremely sharp in definition and can
easily be distinguished for size, shape, and texture. FIG. 6
depicts the same cells on the same slide in FIG. 3 that were
originally stained with the staining solution and subsequently
restained with standard Papanicolaou stain. Note that while the
cellular features are preserved, the nuclear detail is less defined
and more difficult to ascertain. FIG. 7 depicts abnormal cervical
cells that were stained with the staining solution as described
herein and subsequently immobilized onto a microscope slide having
a polycationic charge coating. While the entirety of the cells and
cell groups are readily visualized, the detail of the nucleus is
extremely sharp in definition and can easily be distinguished for
size, shape, and texture. FIG. 8 depicts the same cells on the same
slide in FIG. 3 that were originally stained with the staining
solution and subsequently restained with standard Papanicolaou
stain. Note that while the cellular features are preserved, the
nuclear detail is less defined and more difficult to ascertain.
[0127] As used herein, "about" means within a statistically
meaningful range of a value such as a stated concentration range,
time frame, molecular weight, volume, temperature or pH. Such a
range can be within an order of magnitude, typically within 20%,
more typically still within 10%, and even more typically within 5%
of a given value or range. The allowable variation encompassed by
"about" will depend upon the particular system under study, and can
be readily appreciated by one of skill in the art.
[0128] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
skill in the art to which the invention pertains. Although any
methods and materials similar to or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described
herein.
[0129] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0130] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
* * * * *